PROJECT SUMMARY Human cytomegalovirus (HCMV) infects up to 90% of the U.S. population, resulting in lifelong chronic infection. In immunosuppressed individuals, the outcome is more severe, with lifelong persistent infection associated with various pathologies. HCMV is also a leading viral cause of congenital birth defects. Several antiviral treatments exist, but their use is limited due to significant toxicity, teratogenic effects, poor oral availability and emergence of antiviral resistance. An HCMV vaccine has yet to be licensed. Thus there is a need to understand the mechanism of HCMV infection to establish new targets and approaches for vaccines and therapies. Persistent HCMV infection is complex, involving a balance between productive lytic replication and a silent latent infection involving genome maintenance without replication. The long-term goal is to elucidate mechanisms used by HCMV proteins to control replication and cellular stress responses that influence the fate of infection. Defining these molecular events will augment understanding of the HCMV infected cell and facilitate development of antiviral strategies to manage infection. The experiments presented herein will define the dynamic and antagonistic regulatory relationships between the HCMV kinase pUL97 and pUL27, and determine how this network controls HCMV viral DNA synthesis. HCMV pUL97 regulates pUL27, Tip60 acetyl-transferase and the downstream effector, p21Cip1. The hypothesis is that pUL97 kinase activates Tip60-dependent chromatin remodeling while also inhibiting the pUL27-mediated degradation of Tip60 and the corresponding p21Cip1 expression to promote viral DNA synthesis. This will be tested in three specific aims: Aim 1 Test the hypothesis that pUL27 induces p21Cip1 and prolonged p21Cip1 expression inhibits factors involved in viral DNA synthesis; Aim 2 Test the hypothesis that pUL97 activation of Tip60 promotes viral DNA synthesis in a manner that involves chromatin modifications; and AIM 3 Test the hypothesis that the pUL27-Tip60-pUL97 axis regulates entry into and exit from HCMV latency. A comprehensive repertoire of cellular and molecular biological techniques, viral genetic analysis, transcriptomics, and quantitative ?bottom up? and ?top down? proteomics will be used to study both lytic and latent models of HCMV infection. These studies will define how pUL97 kinase promotes viral DNA synthesis and how the antagonistic activities of two HCMV proteins, pUL97 and pUL27, regulate Tip60 and p21Cip1 expression and control HCMV replication. Understanding this regulatory switch will elucidate the mechanism by which therapeutics may silence HCMV replication.